A detection reagent for detecting escherichia coli and a preparation method thereof

Abstract

The application relates to a detection reagent for detecting escherichia coli and a preparation method, and belongs to the technical field of food safety detection. The detection reagent is a graphene-loaded targeted copper-chlorine co-doped carbon nanometer enzyme aptamer compound, is a light brown transparent liquid, and is prepared by reacting magnetic graphene, (3-dimethylaminopropyl)-3-ethyl carbodiimide, N-hydroxysuccinimide, copper-chlorine co-doped carbon nanometer enzyme and amino-modified aptamer according to a molar mass ratio of 15g:3mol:1.5mol:262.5g:0.3mol. The application carries out cascade signal amplification based on the targeted copper-chlorine co-doped carbon nanometer enzyme with high signal output and the reverse magnetic attraction strategy; when the to-be-detected substance contains escherichia coli O157:H7, the targeted copper-chlorine co-doped carbon nanometer enzyme on the magnetic carrier can specifically recognize the target strain to form a composite structure, the interference of the magnetic carrier on the signal output is eliminated through the reverse magnetic selection effect, the supernatant is taken to measure the fluorescence intensity at 456nm and the ultraviolet absorption intensity at 654nm to determine the concentration of the escherichia coli O157:H7.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a method for preparing a detection reagent for detecting Escherichia coli. Background Technology

[0002] Food contamination by pathogenic bacteria is a major global health problem. *Escherichia coli* O157:H7, a serotype of *E. coli*, is a leading cause of foodborne illnesses. Food is a significant route of contamination for *E. coli* O157:H7, causing serious harm to human health and resulting in substantial losses for the food industry. Therefore, developing highly specific and sensitive detection reagents is a crucial technological prerequisite for the effective prevention and control of *E. coli* O157:H7.

[0003] Currently, detection reagents for *E. coli* include microbial culture media, antibodies with specific recognition elements, and novel aptamer sensors. Traditional culture methods based on microbial culture media require multiple steps, including bacterial isolation and culture, morphological observation, and biochemical identification, which are cumbersome and time-consuming, failing to meet the market demand for rapid testing. Antibodies, as specific recognition elements in immunological methods, are expensive, require large and sophisticated instruments, and are greatly affected by external environmental factors. As for novel biosensor detection technologies based on aptamers, most of them use a single signal output, resulting in significant errors in the detection results. Furthermore, when applied to food testing, complex matrices can reduce sensitivity, making it impossible to detect trace amounts of *E. coli* O157:H7. Therefore, the development of simple, rapid, accurate, and highly sensitive detection reagents for the detection of *E. coli* O157:H7 is urgently needed.

[0004] To shorten detection time, improve detection sensitivity, and reduce detection costs, detection strategies based on catalase activity have been developed and applied. Catalase catalyzes the generation of hydroxyl radicals from hydrogen peroxide, oxidizing the colorless substrate 3,3′,5,5′-tetramethylbenzidine to form a blue product, exhibiting excellent catalytic activity and substrate specificity. However, stringent storage conditions have prompted researchers to develop novel peroxidase mimics with simpler synthesis and more stable structures. Carbon nanozymes, as a novel catalyst, have attracted widespread attention due to their simple synthesis steps and environmental friendliness. Among them, carbon nanozymes using lignin, the second largest renewable resource, as a precursor have been widely favored by researchers. Chloride ions play an important role in the adsorption and reduction of hydrogen peroxide, and their presence can also accelerate the electron loss process from hydrogen peroxide to hydroxyl radicals. However, explorations into the co-doping of chloride ions with other highly catalytically active metal elements have not yet been reported. Furthermore, detection strategies based on nanozymes are limited by low detection sensitivity and weak anti-interference ability of food matrices. There is an urgent need for novel high-sensitivity, low-cost nanozyme detection reagents combined with signal amplification and magnetic separation technologies to achieve ultra-high sensitivity detection of Escherichia coli O157:H7 in food. Summary of the Invention

[0005] To achieve highly sensitive, visual, and rapid detection of Escherichia coli in food matrices, this invention proposes a method for preparing a detection reagent for detecting Escherichia coli.

[0006] A detection reagent for detecting Escherichia coli is a graphene-supported targeted copper-chlorine co-doped carbon nanozyme aptamer complex, which is a light brown transparent liquid.

[0007] The graphene-supported targeted copper-chlorine co-doped carbon nanozyme aptamer complex, with a molar mass ratio of 15 g: 3 mol: 1.5 mol: 262.5 g: 0.3 mol, is prepared by reacting magnetic graphene, (3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, copper-chlorine co-doped carbon nanozyme, and aminated aptamer.

[0008] The copper-chlorine co-doped carbon nanozyme was prepared by reacting sodium lignosulfonate and copper chloride in a mass ratio of 1:1.

[0009] The copper-chlorine co-doped carbon nanozyme and the aminated aptamer are reacted to form a targeted copper-chlorine co-doped carbon nanozyme aptamer.

[0010] The DNA sequence of the aminated aptamer is shown in SEQ ID No:1.

[0011] The preparation steps of a detection reagent for detecting Escherichia coli are as follows:

[0012] (1) Preparation of magnetic graphene

[0013] (1.1) Graphene oxide and sodium hydroxide were mixed in ultrapure water and sonicated to obtain a graphene oxide solution;

[0014] (1.2) Mix ferric salt, ferrous salt and hydrochloric acid to obtain a reaction solution;

[0015] The mass ratio of graphene oxide, iron salt, and ferrous salt is 52:475:250;

[0016] (1.3) The reaction solution was added dropwise to the graphene oxide solution to carry out the reaction, and magnetic graphene with a mass-volume concentration of 150 μg / mL was obtained.

[0017] (2) Preparation of copper-chlorine co-doped carbon nanozymes

[0018] Sodium lignosulfonate and copper chloride were ultrasonically dispersed in deionized water at a mass ratio of 1:1, incubated, cooled to room temperature, and then centrifuged, purified, dialyzed, concentrated and freeze-dried to obtain a copper chloride co-doped carbon nanozyme with a mass volume concentration of 525 μg / mL.

[0019] (3) Preparation of targeted copper-chlorine co-doped carbon nanozyme aptamers

[0020] With a molar mass ratio of 3 mol: 1.5 mol: 262.5 g: 0.3 mol, (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloric acid solution, N-hydroxysuccinimide solution and copper chloride co-doped carbon nanozyme were mixed and then added to amino-modified aptamer solution and incubated to obtain targeted copper chloride co-doped carbon nanozyme aptamer;

[0021] (4) Preparation of detection reagents for detecting Escherichia coli

[0022] 1 mL of targeted copper-chlorine co-doped carbon nanozyme aptamer was added to 100 μL of magnetic graphene with a mass-volume concentration of 150 μg / mL and incubated to obtain a graphene-loaded targeted copper-chlorine co-doped carbon nanozyme aptamer complex, which is the detection reagent for detecting Escherichia coli.

[0023] Further technical solutions are as follows:

[0024] The specific operations in step (1) are as follows:

[0025] (1.1) Take 26 mL of graphene oxide with a mass-volume concentration of 2 mg / mL and a pH value of 8.23, add 1.4 g of sodium hydroxide particles and 5.25 mL of ultrapure water, shake and mix for 10 min, and then sonicate for 30 min to obtain a graphene oxide solution.

[0026] (1.2) Take 0.475 g of ferric chloride and 0.25 g of ferrous chloride, add 0.104 mL of 12 M hydrochloric acid and 3.021 mL of ultrapure water respectively, shake and mix for 1 min to obtain 3.125 mL of reaction solution;

[0027] (1.3) 3.125 mL of the reaction solution was slowly added dropwise to the graphene oxide solution and reacted at 80 °C under nitrogen atmosphere for 1 h to obtain magnetic graphene with a mass-volume concentration of 150 μg / mL.

[0028] The specific operations in step (2) are as follows:

[0029] (2.1) Take 39 mL of deionized water, add 100 mg of sodium lignosulfonate and 100 mg of copper chloride, and sonicate for 10 min to obtain the sonic product;

[0030] (2.2) In a reaction vessel, the ultrasonic product was incubated at 180°C for 8 hours and then naturally cooled to room temperature to obtain a suspension;

[0031] (2.3) The suspension was centrifuged at 6500 rpm for 15 min and purified by filtration through a 0.22 µm membrane to obtain the filtrate;

[0032] (2.4) The filtrate was dialyzed through a 3500 Da dialysis bag for 12 h, collected and concentrated, and then freeze-dried and dialyzed to obtain copper chloride co-doped carbon nanozyme with a mass-volume concentration of 525 μg / mL;

[0033] The specific operations in step (3) are as follows:

[0034] (3.1) Add 300 μL of 10 mM (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloric acid solution and 5 mM N-hydroxysuccinimide solution to 500 μL of 525 μg / mL copper chloride co-doped carbon nanozyme and shake at 150 rpm for 30 min.

[0035] (3.2) Add 1 mL of amino-modified aptamer solution with a molar concentration of 300 nM and incubate at 200 rpm for 3 h to obtain the targeted copper-chlorine co-doped carbon nanozyme aptamer.

[0036] In step (4), the incubation conditions are: 37℃ for 10 min.

[0037] The beneficial technical effects of this invention are reflected in the following aspects:

[0038] (1) This invention synthesizes a non-toxic, environmentally friendly, and highly catalytically active copper-chlorine co-doped lignin carbon nanozyme by mixing lignin with copper and chlorine. The addition of copper can promote a synergistic effect with chlorine, enhancing the activity of the oxidase. The effect is better than that of potassium chloride and hydrochloric acid doping. Its peroxidase activity is increased by 56.6% and 94.0%, respectively, which significantly improves the detection reagents developed based on it.

[0039] (2) This invention utilizes a reverse magnetic separation strategy combined with copper-chlorine co-doped carbon nanozymes with high peroxidase catalytic activity to achieve dual cascade signal amplification, significantly improving the sensitivity of the detection reagent, avoiding detection interference caused by the material itself, and enhancing the specificity of the detection reagent. This provides a new method for the ultra-precise detection of trace Escherichia coli O157:H7 in food. In addition, the aptamer and magnetic nanomaterials are directly adsorbed through π-π stacking, effectively avoiding expensive chemical modifications and reducing detection costs by 30%.

[0040] (3) Verification showed that the detection reagent of the present invention has a high degree of linearity, with a correlation coefficient as high as 0.99821, and exhibits a low detection limit of 57.2 cfu / mL. Current existing technologies generally have detection limits exceeding 10 for Escherichia coli O157:H7. 2 The detection time of this invention is less than 1 hour, while traditional techniques have detection times ranging from 4 hours to 7 days. Furthermore, the dual-modal detection signals of fluorescence and colorimetric methods ensure excellent accuracy even at high sensitivity. The correlation coefficients between the fluorescence intensity and UV absorption intensity values ​​obtained by substituting them into the linear regression equations are 0.99955 and 0.99993, respectively, with the true values ​​of *E. coli* O157:H7 in the sample.

[0041] (4) In summary, the copper-chlorine co-doped carbon nanozyme of this invention is the strongest peroxide nanozyme with lignin as a green raw material. Through reverse magnetic attraction and cooperation with the copper-chlorine co-doped carbon nanozyme with ultra-strong signal output, cascade signal amplification is achieved, which significantly improves the detection speed, accuracy and stability of trace Escherichia coli O157:H7 in food. By specifically reacting with Escherichia coli O157:H7 through the aptamer and separating the magnetic carrier from the signal output system under the action of an external magnetic field, the detection sensitivity and reliability are further improved. In addition, fluorescence and colorimetric signals are detected simultaneously in the test solution. A single experiment can obtain dual-modal signals of fluorescence and colorimetry, which not only enhances the detection sensitivity, but also avoids misjudgment caused by operational errors and food matrix by mutual correction of the two data results. Attached Figure Description

[0042] Figure 1This is a schematic diagram of the detection method of the present invention.

[0043] Figure 2 This is a scanning electron microscope image of Escherichia coli O157:H7 in Example 2 of the present invention.

[0044] Figure 3 This is an elemental mapping diagram of Escherichia coli O157:H7 in Example 2 of the present invention.

[0045] Figure 4 This is a standard curve of the logarithmic concentration of Escherichia coli O157:H7 and the fluorescence intensity value in Example 2 of the present invention.

[0046] Figure 5 This is a standard curve of the logarithmic concentration of Escherichia coli O157:H7 and the absorbance value in Example 2 of the present invention.

[0047] Figure 6 This is a graph showing the specific fluorescence intensity values ​​of the detection method in Example 5 of the present invention.

[0048] Figure 7 This is a graph showing the specific absorbance values ​​of the detection method in Embodiment 5 of the present invention. Detailed Implementation

[0049] The present invention will be further described below with reference to embodiments, but this is not intended to limit the invention.

[0050] Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0051] Unless otherwise specified, the test reagents and consumables used in the following examples are all conventional biochemical reagents; the experimental methods are all conventional methods unless otherwise specified; the quantitative tests in the following examples are all repeated three times, and the results are averaged; unless otherwise specified, the percentages in the following examples are all mass percentages.

[0052] In the following examples, 10× enzyme digestion buffer was purchased from Dalian Takara Bio Inc.; 3,3′,5,5′-tetramethylbenzidine was purchased from Aladdin Reagent Co., Ltd.; 10M hydrogen peroxide solution was purchased from Shanghai Wokai Biotechnology Co., Ltd.; N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were purchased from Sigma-Aldrich, USA; sodium lignosulfonate was purchased from Aladdin Reagent Co., Ltd.; anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd.; copper chloride was purchased from Merck Chemicals Co., Ltd. (Shanghai, China); a 0.22 μm filter membrane was purchased from Jinteng Experimental Equipment Co., Ltd.; and the aminated aptamer was purchased from Shanghai Sangon Biotech Co., Ltd.

[0053] The bacterial strains used in this invention include Escherichia coli O157:H7 (ATCC 43895), Listeria monocytogenes (ATCC 7644), Salmonella typhimurium (ATCC 14028), Pseudomonas aeruginosa (ATCC 25922), Vibrio parahaemolyticus (ATCC 17802), Staphylococcus aureus (ATCC 65389), and Cronobacter sakazakii (ATCC 12806), all provided by the Guangdong Institute of Microbiology.

[0054] All instruments, equipment, raw materials, reagents, and methods used in this invention are ensured to be processed under sterile conditions.

[0055] Any instruments, equipment, raw materials, reagents, or method steps not mentioned in this invention are conventional or well-known techniques to those skilled in the art, and will not be described in detail here.

[0056] Example 1

[0057] The preparation steps of a detection reagent for detecting Escherichia coli are as follows:

[0058] (1) Preparation of magnetic graphene

[0059] (1.1) Take 26 mL of graphene oxide with a mass-volume concentration of 2 mg / mL and a pH value of 8.23, add 1.4 g of sodium hydroxide particles and 5.25 mL of ultrapure water, shake and mix for 10 min, and then sonicate for 30 min to obtain a graphene oxide solution.

[0060] (1.2) Take another 0.475 g of ferric chloride and 0.25 g of ferrous chloride, add 0.104 mL of 12 M hydrochloric acid and 3.021 mL of ultrapure water respectively, shake and mix for 1 min to obtain 3.125 mL of reaction solution;

[0061] (1.3) 3.125 mL of the reaction solution was slowly added dropwise to the above graphene oxide solution, and the reaction was carried out at 80 °C under nitrogen atmosphere for 1 h to obtain magnetic graphene with a mass-volume concentration of 150 μg / mL.

[0062] (2) Preparation of copper-chlorine co-doped carbon nanozymes

[0063] (2.1) Take 39 mL of deionized water, add 100 mg of sodium lignosulfonate and 100 mg of copper chloride, and sonicate for 10 min to obtain the sonic product;

[0064] (2.2) In the reaction vessel, the ultrasonic product was incubated at 180℃ for 8 hours and then naturally cooled to room temperature to obtain a suspension;

[0065] (2.3) The suspension was centrifuged at 6500 rpm for 15 min and purified by filtration through a 0.22 µm membrane to obtain the filtrate;

[0066] (2.4) The filtrate was dialyzed through a 3500 Da dialysis bag for 12 h, collected and concentrated, and then freeze-dried and dialyzed to obtain copper chloride co-doped carbon nanozyme with a mass-volume concentration of 525 μg / mL;

[0067] (3) Preparation of targeted copper-chlorine co-doped carbon nanozyme aptamers

[0068] (3.1) Add 300 μL of 10 mM (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloric acid solution and 5 mM N-hydroxysuccinimide solution to 500 μL of 525 μg / mL copper chloride co-doped carbon nanozyme and shake at 150 rpm for 30 min.

[0069] (3.2) Add 1 mL of amino-modified aptamer solution with a molar concentration of 300 nM and incubate at 200 rpm for 3 h to obtain the targeted copper-chlorine co-doped carbon nanozyme aptamer;

[0070] The DNA sequence of the amino-modified aptamer is shown in SEQ ID No:1, and the 5' end of the modified aptamer is modified with an amino group.

[0071] (4) Preparation of detection reagents for detecting Escherichia coli

[0072] 1 mL of targeted copper-chlorine co-doped carbon nanozyme aptamer was added to 100 μL of magnetic graphene with a volume concentration of 150 μg / mL, and incubated at 37 °C for 10 min to obtain graphene-loaded targeted copper-chlorine co-doped carbon nanozyme aptamer complex, which is the detection reagent for detecting Escherichia coli.

[0073] The detection reagent prepared in Example 1 for detecting Escherichia coli is a light brown transparent liquid.

[0074] Example 2

[0075] Establishment of detection equation

[0076] (1) Take 1 mL of Escherichia coli O157:H7 stock solution cultured in LB broth for 12 h to the late logarithmic growth stage, transfer it to a sterilized centrifuge tube, centrifuge at 5000×g for 5 min, discard the supernatant, and resuspend in 1 mL of 0.1 M, pH 7.4 sterile PBS buffer to obtain a bacterial suspension; use 0.1 M, pH 7.4 sterile PBS buffer to serially dilute the bacterial suspension to prepare a 10% concentration. 1 10 2 103 10 4 10 5 10 6 A cfu / mL Escherichia coli O157:H7 solution was prepared; a 0.1M sterile PBS buffer solution with a pH of 7.4 was prepared as a blank control solution, and the concentration of Escherichia coli O157:H7 in the blank control solution was 0 cfu / mL.

[0077] (2) In 200 μL of blank control solution and concentration of 10 1 10 2 10 3 10 4 10 5 10 6 In a CFU / mL E. coli O157:H7 solution, add 50 μL of the same double-stranded probe solution to each solution, incubate at 37 °C for 60 min, centrifuge at 5000 × g for 5 min, and collect the supernatant to obtain seven composite solutions.

[0078] (3) Seven identical 200 μL graphene-loaded targeted copper chloride co-doped carbon nanozyme aptamer complexes were added to seven 1 mL composite solutions respectively, and the composite solutions were obtained after incubation at 37 °C for 40 min.

[0079] The graphene-supported targeted copper-chlorine co-doped carbon nanozyme aptamer complex was prepared in Example 1;

[0080] (4) Place the seven composite solutions on a magnetic rack and let them stand for 2 minutes. Take the supernatant solution to obtain seven test solutions.

[0081] (5) First, take seven 50 μL supernatants and add them to 450 μL 1× PBS solution. Measure the fluorescence intensity at 456 nm at an excitation wavelength of 330 nm.

[0082] (6) Take seven 50 μL supernatant solutions, add 50 μL of 10 M hydrogen peroxide solution and 50 μL of 3,3′,5,5′-tetramethylbenzidine solution respectively, add 350 μL of acetate-sodium acetate buffer solution, react at 35 °C in the dark for 10 min, and measure the ultraviolet absorption intensity at 654 nm.

[0083] The 100 mM hydrogen peroxide solution was prepared by adding 10 μL of 10 M hydrogen peroxide to 990 μL of ultrapure water; the 4.8 g / L 3,3′,5,5′-tetramethylbenzidine solution was prepared by adding 0.048 g of 3,3′,5,5′-tetramethylbenzidine to 10 mL of anhydrous ethanol.

[0084] (7) Observation revealed that it contained a concentration of 10 2 10 3 10 4 10 5 10 6 The reaction solution of CFU / mL E. coli O157:H7 turns blue, indicating a positive result; the solution containing a blank control and a concentration of 10... 1 The reaction solution of CFU / mL E. coli O157:H7 was colorless and transparent, and the result was negative. Therefore, the visual detection limit is 10. 2 The standard curve was plotted with the fluorescence intensity of the five positive reaction solutions at 456 nm as the ordinate (Y) and the logarithm of the *E. coli* O157:H7 bacterial concentration (cfu / mL) as the abscissa (X). The results are shown in Appendix 1. Figure 4 The detection equation was calculated as: Y = 0.14843X - 0.06199, with a correlation coefficient of 0.99821. Five 200μL positive reaction solutions were used, with the absorbance intensity at 654nm peaking as the ordinate (Y). A standard curve (2) was plotted with the logarithm of the *E. coli* O157:H7 bacterial concentration (cfu / mL) as the abscissa (X). The results are shown in the appendix. Figure 5 The detection equation is: Y = 0.26039X - 0.32652, and the correlation coefficient is 0.99345.

[0085] Example 3

[0086] Application of this invention in detecting the content of Escherichia coli O157:H7 in ham

[0087] (1) Sample processing

[0088] The ham to be tested was cut into 25g pieces and mixed with 25mL of 0.1M PBS solution (pH 7.4) in a sterilization bag. The mixture was homogenized for 2 minutes to obtain a PBS mixture. Then, 3×10⁻⁶ ppm of the PBS solution was added. 4 CFU / mL Escherichia coli O157:H7 solution, let the spiked mixture stand for 5 min, take the supernatant to obtain the test solution;

[0089] (2) Detection

[0090] (2.1) Take 200 μL of graphene-supported targeted copper chloride co-doped carbon nanozyme aptamer complex and add 1 mL of test solution. After incubating at 37 °C for 40 min, the composite solution is obtained.

[0091] The graphene-supported targeted copper-chlorine co-doped carbon nanozyme aptamer complex was obtained from Example 1;

[0092] (2.2) Perform magnetic separation on the composite solution. After standing on the magnetic rack for 2 minutes, take the supernatant solution to obtain the test solution;

[0093] (2.3) Take 50 μL of the above test solution and add it to 450 μL of 1×PBS solution. Measure the fluorescence intensity at 456 nm at an excitation wavelength of 330 nm.

[0094] (2.4) Take another 50 μL of the above test solution, add 50 μL of 100 mM hydrogen peroxide solution and 50 μL of 4.8 g / L tetramethylbenzidine solution respectively, add 350 μL of acetate-sodium acetate buffer solution, react at 35 °C in the dark for 10 min, and measure its ultraviolet absorption intensity at 654 nm;

[0095] (3) Calculate the test results

[0096] (3.1) Observe the color of the reaction solution. When the reaction solution is colorless and transparent, the test result is negative; when the reaction solution is blue, the test result is positive. Substitute the fluorescence intensity value I of the reaction solution of the positive sample at 456 nm into the detection equation. The detection equation is: Y = 0.14843X - 0.06199. In the equation, X is the logarithm of the concentration of Escherichia coli O157:H7, Y is I, and I is the fluorescence intensity value at 456 nm when the test solution is detected by this invention. Calculate the concentration of Escherichia coli O157:H7 in the test solution, and the detection is complete. Take another 200 μL of the positive reaction solution and measure its ultraviolet absorption intensity value A at 654 nm. Substitute it into the standard curve 2. The detection equation is Y = 0.26039X - 0.32652. Calculate the concentration of Escherichia coli O157:H7 in the test solution and calibrate it with the target bacterial concentration obtained from the fluorescence.

[0097] The detection equation was obtained from Example 2;

[0098] (3.2) Observation revealed that the reaction solution was blue, indicating a positive result. 200 μL of the positive reaction solution was placed in a cuvette, and its fluorescence value at 456 nm was measured to be 0.599. Substituting this value into detection equation 1, the concentration of *E. coli* O157:H7 in the analyte was calculated to be 2.84 × 10⁻⁶. 4 CFU / mL; A separate 200 μL positive reaction solution was taken, and its UV absorbance at 654 nm was measured to be 0.843, indicating that the concentration of *E. coli* O157:H7 in the analyte was 3.09 × 10⁻⁶. 4 cfu / mL.

[0099] Example 4

[0100] Application of this invention in detecting the content of Escherichia coli O157:H7 in milk

[0101] (1) Sample processing

[0102] Take 1 mL of commercially available milk, centrifuge at 5000×g for 5 min, discard the supernatant, and resuspend the precipitate in 1 mL of 0.1 M, pH 7.4 sterile PBS buffer to obtain the test solution.

[0103] (2) Detection

[0104] (2.1) Take 200 μL of graphene-supported targeted copper chloride co-doped carbon nanozyme aptamer complex and add 1 mL of test solution. After incubating at 37 °C for 40 min, the composite solution is obtained.

[0105] The graphene-supported targeted copper-chlorine co-doped carbon nanozyme aptamer complex was obtained from Example 1;

[0106] (2.2) Perform magnetic separation on the composite solution. After standing on the magnetic rack for 2 minutes, take the supernatant solution to obtain the test solution;

[0107] (2.3) Take 50 μL of the above test solution and add it to 450 μL of 1×PBS solution. Measure the fluorescence intensity at 456 nm at an excitation wavelength of 330 nm.

[0108] (2.4) Take another 50 μL of the above test solution, add 50 μL of 100 mM hydrogen peroxide solution and 50 μL of 4.8 g / L tetramethylbenzidine solution respectively, add 350 μL of acetate-sodium acetate buffer solution, react at 35 °C in the dark for 10 min, and measure its ultraviolet absorption intensity at 654 nm;

[0109] (3) Calculate the test results

[0110] (3.1) Observe the color of the reaction solution. When the reaction solution is colorless and transparent, the test result is negative; when the reaction solution is blue, the test result is positive. Substitute the fluorescence intensity value I of the reaction solution of the positive sample at 456 nm into the detection equation. The detection equation is: Y = 0.14843X - 0.06199. In the equation, X is the logarithm of the concentration of Escherichia coli O157:H7, Y is I, and I is the fluorescence intensity value at 456 nm when the test solution is detected by this invention. Calculate the concentration of Escherichia coli O157:H7 in the test solution, and the detection is complete. Take another 200 μL of the positive reaction solution and measure its ultraviolet absorption intensity value A at 654 nm. Substitute it into the standard curve 2 to calculate the concentration of Escherichia coli O157:H7 in the test solution. Calibrate it with the target bacterial concentration obtained from the fluorescence.

[0111] The detection equation was obtained from Example 2;

[0112] (3.2) Observation revealed that the reaction solution was transparent and the test result was negative, indicating that Escherichia coli O157:H7 was not detected in the test sample.

[0113] Example 5

[0114] Application of this invention in detecting the content of Escherichia coli O157:H7 in drinking water

[0115] (1) Sample processing

[0116] Drinking water was used directly as the test solution without any treatment.

[0117] (2) Detection

[0118] (2.1) Take 200 μL of graphene-supported targeted copper chloride co-doped carbon nanozyme aptamer complex and add 1 mL of test solution. After incubating at 37 °C for 40 min, the composite solution is obtained.

[0119] The graphene-supported targeted copper-chlorine co-doped carbon nanozyme aptamer complex was obtained from Example 1;

[0120] (2.2) Perform magnetic separation on the composite solution. After standing on the magnetic rack for 2 minutes, take the supernatant solution to obtain the test solution;

[0121] (2.3) Take 50 μL of the above test solution and add it to 450 μL of 1×PBS solution. Measure the fluorescence intensity at 456 nm at an excitation wavelength of 330 nm.

[0122] (2.4) Take another 50 μL of the above test solution, add 50 μL of 100 mM hydrogen peroxide solution and 50 μL of 4.8 g / L tetramethylbenzidine solution respectively, add 350 μL of acetate-sodium acetate buffer solution, react at 35 °C in the dark for 10 min, and measure its ultraviolet absorption intensity at 654 nm;

[0123] (3) Calculate the test results

[0124] (3.1) Observe the color of the reaction solution. When the reaction solution is colorless and transparent, the test result is negative; when the reaction solution is blue, the test result is positive. Substitute the fluorescence intensity value I of the reaction solution of the positive sample at 456 nm into the detection equation. The detection equation is: Y = 0.14843X - 0.06199. In the equation, X is the logarithm of the concentration of Escherichia coli O157:H7, Y is I, and I is the fluorescence intensity value at 456 nm when the test solution is detected by this invention. Calculate the concentration of Escherichia coli O157:H7 in the test solution, and the detection is complete. Take another 200 μL of the positive reaction solution and measure its ultraviolet absorption intensity value A at 654 nm. Substitute it into the standard curve 2 to calculate the concentration of Escherichia coli O157:H7 in the test solution. Calibrate it with the target bacterial concentration obtained from the fluorescence.

[0125] The detection equation was obtained from Example 2;

[0126] (3.2) Observation revealed that the reaction solution was transparent and the test result was negative, indicating that Escherichia coli O157:H7 was not detected in the test sample.

[0127] Example 6

[0128] Specific detection method for Escherichia coli O157:H7

[0129] (1) Sample processing

[0130] Take seven sterile centrifuge tubes and add 1 mL of sterile PBS buffer (control) and PBS buffer (both with a concentration of 10) to each tube respectively. 6 Seven mixtures were prepared by collecting cfu / mL Escherichia coli O157:H7, Salmonella typhimurium, Pseudomonas aeruginosa, Cronobacter, Staphylococcus aureus, and Vibrio parahaemolyticus. The seven mixtures were centrifuged at 5000×g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1 mL of 0.1M, pH 7.4 sterile PBS buffer to obtain seven test solutions.

[0131] (2) Detection

[0132] (2.1) Take 200 μL of graphene-supported targeted copper chloride co-doped carbon nanozyme aptamer complex and add 1 mL of test solution. After incubating at 37 °C for 40 min, the composite solution is obtained.

[0133] The graphene-supported targeted copper-chlorine co-doped carbon nanozyme aptamer complex was obtained from Example 1;

[0134] (2.2) Perform magnetic separation on the composite solution. After standing on the magnetic rack for 2 minutes, take the supernatant solution to obtain the test solution;

[0135] (2.3) Take 50 μL of the above test solution and add it to 450 μL of 1×PBS solution. Measure the fluorescence intensity at 456 nm at an excitation wavelength of 330 nm.

[0136] (2.4) Take another 50 μL of the above test solution, add 50 μL of 100 mM hydrogen peroxide solution and 50 μL of 4.8 g / L tetramethylbenzidine solution respectively, add 350 μL of acetate-sodium acetate buffer solution, react at 35 °C in the dark for 10 min, and measure its ultraviolet absorption intensity at 654 nm;

[0137] (3) Calculate the test results

[0138] (3.1) Observe the color of the reaction solution. When the reaction solution is colorless and transparent, the test result is negative; when the reaction solution is blue, the test result is positive. Substitute the fluorescence intensity value I of the reaction solution of the positive sample at 456 nm into the detection equation. The detection equation is: Y = 0.14843X - 0.06199. In the equation, X is the logarithm of the concentration of Escherichia coli O157:H7, Y is I, and I is the fluorescence intensity at 456 nm when the test solution is detected by this invention. Calculate the concentration of Escherichia coli O157:H7 in the test solution, and the detection is complete. Take another 200 μL of the positive reaction solution and measure its ultraviolet absorption intensity value A at 654 nm. Substitute it into the standard curve 2 to calculate the concentration of Escherichia coli O157:H7 in the test solution. Calibrate it with the target bacterial concentration obtained from the fluorescence.

[0139] The detection equation was obtained from Example 2;

[0140] Observation revealed that the reaction solution containing *E. coli* O157:H7 was blue, indicating a positive test result; the reaction solutions containing sterile PBS solution (control), *Salmonella typhimurium*, *Listeria monocytogenes*, *Pseudomonas aeruginosa*, *Cronobacter*, *Staphylococcus aureus*, and *Vibrio parahaemolyticus* were colorless and transparent, indicating negative test results; see attached. Figure 6 As shown, the fluorescence intensity values ​​of the seven reaction solutions measured at 456 nm were as follows: Escherichia coli O157:H7 0.9876; PBS solution 0.20326; Salmonella typhimurium 0.2790; Pseudomonas aeruginosa 0.1987; Cronobacterium tumefaciens 0.1979; Staphylococcus aureus 0.2114; Vibrio parahaemolyticus 0.2096; as shown in the attached figure. Figure 7As shown, the UV absorption intensities of the seven reaction solutions at 654 nm were as follows: Escherichia coli O157:H7 1.1937; PBS solution 0.1142; Salmonella typhimurium 0.1275; Pseudomonas aeruginosa 0.1047; Cronobacterium 0.0975; Staphylococcus aureus 0.1142; Vibrio parahaemolyticus 0.1123. This demonstrates that the present invention has good specificity for detecting Escherichia coli O157:H7.

Claims

1. A method for preparing a detection reagent for detecting Escherichia coli, characterized by, The operation steps are as follows: (1) Preparation of magnetic graphene (1.1) Take 26 mL of graphene oxide with a mass-volume concentration of 2 mg / mL and a pH value of 8.23, add 1.4 g of sodium hydroxide particles and 5.25 mL of ultrapure water, shake and mix for 10 min, and then sonicate for 30 min to obtain a graphene oxide solution. (1.2) Take 0.475g of ferric chloride and 0.25g of ferrous chloride, add 0.104mL of 12M hydrochloric acid and 3.021mL of ultrapure water respectively, shake and mix for 1min to obtain 3.125mL of reaction solution; (1.3) 3.125 mL of the reaction solution was slowly added dropwise to the above graphene oxide solution, and the reaction was carried out at 80 °C under nitrogen atmosphere for 1 h to obtain magnetic graphene with a mass-volume concentration of 150 μg / mL. (2) Preparation of copper-chlorine co-doped carbon nanozymes (2.1) Take 39 mL of deionized water, add 100 mg of sodium lignosulfonate and 100 mg of copper chloride, and sonicate for 10 min to obtain the sonic product; (2.2) In the reaction vessel, the ultrasonic product was incubated at 180℃ for 8 hours and then naturally cooled to room temperature to obtain a suspension; (2.3) The suspension was centrifuged at 6500 rpm for 15 min and purified by filtration through a 0.22 µm membrane to obtain the filtrate; (2.4) The filtrate was dialyzed through a 3500 Da dialysis bag for 12 h, collected and concentrated, and then freeze-dried and dialyzed to obtain copper chloride co-doped carbon nanozyme with a mass-volume concentration of 525 μg / mL; (3) Preparation of targeted copper-chlorine co-doped carbon nanozyme aptamers (3.1) Add 300 μL of 10 mM (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloric acid solution and 5 mM N-hydroxysuccinimide solution to 500 μL of 525 μg / mL copper chloride co-doped carbon nanozyme and shake at 150 rpm for 30 min. (3.2) Add 1 mL of amino-modified aptamer solution with a molar concentration of 300 nM and incubate at 200 rpm for 3 h to obtain the targeted copper-chlorine co-doped carbon nanozyme aptamer; The DNA sequence of the amino-modified aptamer is shown in SEQ ID No:1, and the 5' end of the modified aptamer is modified with an amino group. (4) Preparation of detection reagents for detecting Escherichia coli 1 mL of targeted copper-chlorine co-doped carbon nanozyme aptamer was added to magnetic graphene with a mass-volume concentration of 150 μg / mL and incubated for 10 min to obtain a graphene-loaded targeted copper-chlorine co-doped carbon nanozyme aptamer complex, which is the detection reagent for detecting Escherichia coli.

2. A detection reagent for detecting Escherichia coli, characterized by comprising, The detection reagent is prepared by the preparation method described in claim 1.

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